The single-disulphide intermediates in the refolding of reduced pancreatic trypsin inhibitor

The intermediate species with one disulphide bond in the renaturation of reduced pancreatic trypsin inhibitor have been trapped, isolated, and the Cys residues involved in the disulphide bonds determined. Approximately half the intermediate species had the disulphide bond between Cys-30 and 51, a disulphide bond also present in the native inhibitor. The next most predominant species, representing one-quarter of the total, had a disulphide bond between Cys-5 and 30, and two more minor species involving Cys-30 and 55 and Cys-5 and 51 were detected; these disulphide bonds are not present in the native inhibitor.The nature of the disulphide bonds present are concluded to reflect primarily the conformational forces acting at this stage of folding, which may be primarily interactions between segments with propensities for secondary structure, either helices or β-sheet. The general importance of such interactions in protein folding is discussed.     

On the relevance of non-random polypeptide conformations for protein folding

The possibility that any non-random conformation in reduced bovine pancreatic trypsin inhibitor (BPTI) and ribonuclease A might be significant for folding has been considered, using the experimental data available on forming the first disulphide bond in each. It is a thermodynamic necessity that whatever conformation stabilises a particular disulphide bond be stabilised to the same extent by the presence of the disulphide. The stabilising effects of disulphides are known approximately, so the stability of any non-random conformation found in a one-disulphide intermediate can be estimated in the absence of the disulphide bond. The non-random conformation in the BPTI intermediates is sufficiently labile to indicate that it would be expected to be present in no more than 5% of the reduced BPTI molecules. There is much less non-random conformation apparent in ribonuclease A. Whatever conformations are represented in the bulk of these two reduced proteins cannot favour disulphide formation and further productive folding.     

Analysis of disulphide bond connectivity patterns in protein tertiary structure

The analysis of disulphide bond containing proteins in the Protein Data Bank (PDB) revealed that out of 27,209 protein structures analyzed, 12,832 proteins contain at least one intra-chain disulphide bond and 811 proteins contain at least one inter-chain disulphide bond. The intra-chain disulphide bond containing proteins can be grouped into 256 categories based on the number of disulphide bonds and the disulphide bond connectivity patterns (DBCPs) that were generated according to the position of half-cystine residues along the protein chain. The PDB entries corresponding to these 256 categories represent 509 unique SCOP superfamilies. A simple web-based computational tool is made freely available at the website http://www.ccmb.res.in/bioinfo/dsbcp that allows flexible queries to be made on the database in order to retrieve useful information on the disulphide bond containing proteins in the PDB. The database is useful to identify the different SCOP superfamilies associated with a particular disulphide bond connectivity pattern or vice versa. It is possible to define a query based either on a single field or a combination of the following fields, i.e., PDB code, protein name, SCOP superfamily name, number of disulphide bonds, disulphide bond connectivity pattern and the number of amino acid residues in a protein chain and retrieve information that match the criterion. Thereby, the database may be useful to select suitable protein structural templates in order to model the more distantly related protein homologs/analogs using the comparative modeling methods.     

Presence of new disulphide-bonded collagens in shark Prionace glauca muscle

• 1.1. Two new collagenous fragments were detected in great blue shark myocommata.
• 2.2. The fragments, isolated from pepsin digests, were shown to be sensitive to disulphide bond cleaving agents. The higher molecular weight fragment (designated HMW) of about 250 kDa gave rise to a 40 kDa fragment following reduction. The second fragment with a molecular weight of about 53 kDa (designated LMW) produced a major fragment of about 29 kDa after reduction with disulphide bond cleaving agents.
• 3.3. Type I collagen, a type I-like collagen and type V collagen were also detected in the myocommata.

     

Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe.

1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2'-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2'-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2'-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

Extracellular conserved cysteine forms an intersubunit disulphide bridge in the KCNK5 (TASK-2) K+ channel without having an essential effect upon activity

The functional channel unit of K(+) channels with two pore regions in tandem is thought to be a homodimer and it has been suggested that this dimeric structure occurs by interaction of an extracellular domain, the self-interacting domain. Interaction and functional assembly have been studied in some detail for KCNK1. It is proposed that a disulphide bond between highly conserved C69 residues of the self-interacting domain is formed which is essential for channel activity. We mutated C51, the equivalent residue in the pH-dependent KCNK5, to study its effect on channel function. Western analysis of proteins from cells expressing epitope-tagged KCNK5 and KCNK5-C51S was consistent with reduction-sensitive self-association of monomers dependent upon the presence of C51. Patch-clamp analysis of heterologously expressed KCNK5-C51S, however, revealed it was functional and indistinguishable in rectification properties and pH dependence from the non-mutated channel. The same result was found with KCNK5-C115S. It is concluded that the proposed disulphide bond between cysteine 51 residues of KCNK5 subunits does occur and preserves a dimeric structure in the detergent solubilized complex. Functional assays, on the other hand, suggest that such a disulphide bridge is not essential for correct functional expression.     

Extracellular conserved cysteine forms an intersubunit disulphide bridge in the KCNK5 (TASK-2) K + channel without having an essential effect upon activity

The functional channel unit of K + channels with two pore regions in tandem is thought to be a homodimer and it has been suggested that this dimeric structure occurs by interaction of an extracellular domain, the self-interacting domain. Interaction and functional assembly have been studied in some detail for KCNK1. It is proposed that a disulphide bond between highly conserved C69 residues of the self-interacting domain is formed which is essential for channel activity. We mutated C51, the equivalent residue in the pH-dependent KCNK5, to study its effect on channel function. Western analysis of proteins from cells expressing epitope-tagged KCNK5 and KCNK5-C51S was consistent with reduction-sensitive self-association of monomers dependent upon the presence of C51. Patch-clamp analysis of heterologously expressed KCNK5-C51S, however, revealed it was functional and indistinguishable in rectification properties and pH dependence from the non-mutated channel. The same result was found with KCNK5-C115S. It is concluded that the proposed disulphide bond between cysteine 51 residues of KCNK5 subunits does occur and preserves a dimeric structure in the detergent solubilized complex. Functional assays, on the other hand, suggest that such a disulphide bridge is not essential for correct functional expression.     

Thiol and disulphide contents of hen ovalbumin. C-Terminal sequence and location of disulphide bond

1. The thiol and disulphide contents of hen ovalbumin were investigated by p-chloromercuribenzoate titration, by determination of cysteic acid content after performic acid oxidation, by measurement of uptake of radioactive iodoacetic acid, and by assay of S-aminoethylcysteine after reaction with ethyleneimine. All results showed that ovalbumin had 6 half-cystine residues. Experiments with and without reducing agents demonstrated that there were 4 thiol groups and 1 disulphide bond. 2. A peptide containing equimolar amounts of S-carboxymethyl-cysteine, serine, valine and proline, but no lysine or arginine, was obtained by radioactive labelling of the cysteine residues with iodo[¹⁴C]acetic acid followed by electrophoretic and chromatographic separation of tryptic digests. It was concluded that the C-terminal sequence of ovalbumin is -Cys-Val-Ser-Pro. 3. The location of the disulphide bond was studied by using a double-labelling technique. It was shown that one end of the disulphide was located in this C-terminal peptide.     

Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.     

The heavy chain of tetanus toxin can mediate the entry of cytotoxic gelonin into intact cells

An artificial conjugate of the heavy chain of tetanus toxin linked by a disulphide bond to the impermeant ribosome-inactivating protein gelonin is cytotoxic to intact HT29 cells by inhibiting intracellular protein synthesis. Neither toxin nor gelonin alone has any significant effect. This shows that the heavy chain has the ability to mediate internalization of a protein to which it is bound by a disulphide bond. Thus the normal role of the tetanus toxin heavy chain may be to allow entry of the light chain into a cell.     

The role of the available sulphydryl group in liver butyryl-coenzyme A synthetase

Butyryl-CoA synthetase from acetone-dried ox liver mitochondria can be activated up to 80% by low concentrations of iodoacetate with the concomitant modification of one available enzyme thiol group. The relative molecular mass of the carboxymethylated enzyme was unchanged, and there were no major changes in the complex kinetic properties of the enzyme. Extraction experiments on liver mitochondria indicate that in vivo the enzyme is linked to a mitochondrial membrane by a bond cleavable by disulphide bond reducing agents.     

The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme

Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild-type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

Intramolecular disulphide bond arrangements in nonhomologous proteins

The presence and location of intramolecular disulphide bonds are a key determinant of the structure and function of proteins. Intramolecular disulphide bonds in proteins have previously been analyzed under the assumption that there is no clear relationship between disulphide arrangement and disulphide concentration. To investigate this, a set of sequence nonhomologous protein chains containing one or more intramolecular disulphide bonds was extracted from the Protein Data Bank, and the arrangements of the bonds, Protein Data Bank header, and Structural Characterization of Proteins fold were analyzed as a function of intramolecular disulphide bond concentration. Two populations of intramolecular disulphide bond-containing proteins were identified, with a naturally occurring partition at 25 residues per bond. These populations were named intramolecular disulphide bond-rich and -poor. Benefits of partitioning were illustrated by three results: (1) rich chains most frequently contained three disulphides, explaining the plateaux in extant disulphide frequency distributions; (2) a positive relationship between median chain length and the number of disulphides, only seen when the data were partitioned; and (3) the most common bonding pattern for chains with three disulphide bonds was based on the most common for two, only when the data were partitioned. The two populations had different headers, folds, bond arrangements, and chain lengths. Associations between IDSB concentration, IDSB bonding pattern, loop sizes, SCOP fold, and PDB header were also found. From this, we found that intramolecular disulphide bond-rich and -poor proteins follow different bonding rules, and must be considered separately to generate meaningful models of bond formation.     

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

The positions of the disulphide bridges of the 1,4-beta-glucan cellobiohydrolase (CBH I) of the fungus Trichoderma reesei have been investigated. The results can be summarized as follows. (1) The enzyme contains 12 disulphide bridges and no free cysteine residues. (2) The location of six disulphide bridges have been determined experimentally. (3) The bonding patterns of the two disulphide bridges in the C-terminal region is suggested on the basis of internal homology. (4) The remaining four disulphide bridges are put into two groups, each containing four half-cystine residues where two are adjacent. (5) A repeating bonding pattern is observed along the peptide chain and a non-local disulphide bond with an unusually long separation distance links the N-terminal and the C-terminal region. (6) The disulphide-bonded CNBr peptides of a 1,4-beta-glucan glucanohydrolase (endoglucanase II) from T. reesei have been isolated and a disulphide bonding pattern is suggested on the basis of the sequence homology between the two enzymes.     

Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme. The effects on folding, stability and enzymatic properties.

Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in disulphide bridges. These PLA2s have been classified into several groups based on the disulphide bond patterns found [Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2]. To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2. Refolding experiments indicate that small structural variations play an important role in the folding of newly made PLA2 analogues. Introduction of a C-terminal extension together with disulphide bridge 50-131 gives rise to an enzyme that displays full enzymatic activity having increased conformational stability. In contrast, introduction of a small insertion between positions 88 and 89 together with disulphide bridge 86-89 decreases the catalytic activity significantly, but does not change the stability. Both disulphide bridges 11-77 and 61-91 are important for the kinetic properties and stability of the enzyme. Disulphide bridge 11-77, but not 61-91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2.     

Bacterial conjugation: a two-step mechanism for DNA transport

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli, DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.     

Effect of acetylcholine and acetylcholinesterase on the activity of contractile vacuole of Amoeba proteus

Acetylcholine (ACh, 1 microM) stimulates activity of the contractile vacuole of proteus. The effect of ACh is not mimicked by its analogs which are not hydrolyzed by acetylcholinesterase (AChE), i. e., carbacholine and 5-methylfurmethide. The effect of ACh is not sensitive to the blocking action of M-cholinolytics, atropine and mytolone, but is suppressed by N-cholinolytic, tubocurarine. The inhibitors of AChE, eserine (0.01 microM) and armine (0.1 microM), suppress the effect of ACh on amoeba contractile vacuole. ACh does not affect activation of contractile vacuole induced by arginine-vasopressin (1 microM), but it blocks such effect of opiate receptors agonist, dynorphin A1-13 (0.01 microM). This effect of ACh is also suppressed by the inhibitors of AChE. These results suggest that, in the above-described effects of ACh, AChE acts not as an antagonist, but rather as a synergist.